Rubella was first described in Germany in the 18th century and is, therefore, often referred to as German measles. It is a highly contagious disease characterized by a general rash and a mild fever. Its clinical aspects were, for a long time, confused with other infections, including measles. The major risk associated with rubella infection occurs during the first trimester of pregnancy when severe damage to the fetus can result in deafness, cataracts, cardiac abnormalities and microencephaly.
The rubella virus, the etiologic agent of rubella, belongs to the Togaviridae family. It is a roughly spherical enveloped virus about 60 nm in diameter. Its genome consists of a single positive stranded RNA (10 Kb). The structural polyprotein encoded by this genome consists of two envelope glycoproteins--E1 (58K) and E2(42-47K)--and a nucleocapsid protein--C(33K)--. The viral envelope includes components from the host infected cell membrane, and the two viral glycoproteins E1 and E2. These envelope glycoproteins are responsible for the hemagglutination activity of the rubella virus. E1 and E2 glycoproteins are linked by disulfide bonds to form homo- and heterodimers.
Three strains of rubella virus (Therien, Judith, M33) have been described and portions of their genomes have been sequenced (Frey et al., 1986, Virology 154, 228-232; Terry et al., 1988, Arch. Virol. 98, 189-197; Clarke et al., 1987, Nucl. Acids Res. 15, 3041-3057; Takkinen et al., 1988, J. Gen. Virol. 69, 603-612). The sequences of rubella vaccine strain (RA 27/3) is also known (Nakhasi et al., 1989, Nucl. Acids Res. 17 (11), 4393-4394).
Although rubella can be diagnosed by inoculating infected materials (usually nasopharyngeal secretions) into susceptible cell cultures, the most widely used diagnostic tests are based on the hemagglutinating properties of its glycoproteins. In those assays ("HAI"), the presence in a serum sample of antibodies to the hemagglutinin prevents the virus from binding to red blood cells (usually from chicken blood) thus inhibiting hemagglutination (Peetermans and Huygelen, 1967, Presse Med. 75, 2177-2178 and Lennette and Schmidt, 1979, in "Diagnostic procedures for viral, rickettsial and chlamydial infections", 5th Ed. American Public Health Association Inc., Washington). In such assays an increase in HAI antibody titers is an indication of a recent infection.
Since the introduction of enzyme-labeled antibodies (Avrameas, 1969, Immunochemistry 6, 43-52), enzyme immunoassay (EIA) or enzyme-linked-immunosorbent assays (ELISA) have been used for the diagnosis of a large variety of viral and bacterial infections, including rubella infections. Serodiagnosis of rubella infections using ELISA techniques was first described by Voller and Bidwell (1975, Br. J. Exp. Pathol. 56, 338-339).
In ELISA in particular, viral extracts or lysates are typically coated onto the surface of plastic wells so that antibodies (if present) in a serum sample or analyte will bind to the adsorbed proteins from the viral extracts. After appropriate washing, the presence of antibodies bound to the proteins in the well is detected using antibodies to human immunoglobulins conjugated to an enzyme, such as horseradish peroxidase. The level of enzymatic activity is measured in each well after washing off the unbound enzyme. Other forms and variations of the ELISA test are also well known and often used by persons skilled in the art.
The introduction of ELISA for the specific determination of rubella IgM and IgG has been responsible for the rapid decline (i.e., from 45% in 1978-1980 to 19% in 1982) of HAI diagnostic assays for rubella viral infections (Steece et al., 1985, J. Clin. Microbiol. 21 (1), 140-142). Compared to HAI tests, ELISA requires no sera pre treatment and only one or two dilutions per serum sample. The amount of antigenic material used in ELISA is also smaller than what was required in the former HAI assays.
There are several problems with the presently used ELISA diagnostic tests for rubella infection. Variations between different preparations of rubella antigens used to coat the wells are often observed. These variations are likely a consequence of various difficulties encountered in reproducibly isolating the rubella virus. In tissue culture, the virus grows to low titers, is difficult to separate from cellular membrane debris, and is highly labile (Ho-Terry et al., 1986, Arch. Virol. 87, 219-228; Chagnon and LaFlamme, 1964, Can. J. Microbiol. 10, 501-503). This makes it difficult to isolate the virus from cellular debris originating from host cells. In an attempt to overcome this problem, some ELISA techniques for detecting rubella infection use a series of wells coated with extracts prepared with uninfected cells (reference antigens) and a second series of wells coated with extracts prepared from rubella-infected cells (viral antigens). Each serum sample is then tested on both series of wells and the net response is calculated by subtraction of the signal measured on the reference antigens' wells from the one measured on the viral antigens' wells.
Terry et al. (1988, Arch. Virol. 98, 189-197) and Ho-Terry et al. (1986, Arch. Virol. 90, 145-152 and European Patent Application No. 88306191.3) refer to the reactivity of three non-competing monoclonal antibodies directed against the rubella E1 glycoprotein. The epitopes bound by each of these monoclonal antibodies have been identified and designated EP1, EP2 and EP3. Monoclonal antibodies directed to EP1 and EP2 exhibit both hemagglutination inhibition and neutralizing activity. Monoclonal antibodies to EP3 exhibit only neutralizing activity. Synthetic peptides corresponding to the EP1, EP2 or EP3 epitopes have not been reported to date. The location of these three epitopes in the viral genome has been described in Terry et al. (supra).
Lozzi et al. (1990, Arch. Virol. 110, 271-276) have synthesized overlapping octapeptides covering the region between amino acids 243-286 which includes the EP1, EP2, and EP3 epitopes of the E1 glycoprotein. Their goal was to establish the minimal size of each of these epitopes. They tested each octapeptide with a pool of human high titer anti-rubella IgG isolated from subjects hyperimmunized with rubella vaccine (the authors stressed that this pool contained no less that 400 International Units of anti-rubella immunoglobulins/mL and that the HA titer was about 2000 Units/mL). Using this very concentrated anti-rubella preparation, they have detected low reactivity with all of their peptides. Using rubella positive sera from vaccinated and naturally infected patients, they report that they observed a higher background absorbance than when purified and concentrated immunoglobulins were used. This study illustrates the difficulty one faces when trying to identify synthetic peptide antigens of significant use in the design of a diagnostic test or a better vaccine.
More recently, Wolinsky et al. (1991, J. Virol. 65, 3986-3994) have characterized a series of murine monoclonal antibodies reacting with various regions of the E1 and E2 proteins. Using various plasmid constructs, the authors have localized the binding sites of their monoclonal antibodies. Most of the anti-E1 reactivity was located between residue 202 and 283. On E2, the monoclonal antibodies were binding to a relatively large region covering 116 residues at the amino terminus.
The rubella pandemic of 1963-1965 prompted the development of a vaccine against rubella (Parkman et al., 1966, N. Engl. J. Med. 275, 569-574). It is comprised of live attenuated viruses and is immunogenic in at least 95% of the recipients. Neutralizing antibodies generated by the attenuated vaccine appear later than those following a natural infection and at levels as much as ten- fold lower. Vaccine-induced antibodies, nonetheless, effectively protect recipients from the disease. The present rubella vaccines, however, have some drawbacks. For example, a significant proportion of people vaccinated suffer occasional arthritis (mainly seen in adult women), mild rash, fever and lymphadenopathy. Protection conferred by the vaccine also lasts for only 2-10 years, rather than the longer-lasting immunity that follows natural infection. Most importantly, small amounts of infectious virus typically appear in the nasopharynx 2-3 weeks after immunization, making vaccination very dangerous for pregnant women coming in close contact with a recently vaccinated person or even worse having herself been vaccinated while not knowing she was pregnant.
Vaccines based on synthetic or recombinant peptides would not present this hazard because the antigenic material would be significantly less allergenic or non-allergenic. However, such vaccines are not now available and the immunogenicity and neutralizing properties of peptide-based vaccines are unknown. Furthermore, not all peptides are expected to be useful in vaccines. For example, high antibody titers in HAI tests do not correlate well with protection against rubella infection (Partridge et al., 1981, Br. Med. J. 282, 187-188). This may be due to the fact that epitopes involved in hemagglutination and neutralization are different (Trudel et al., 1982, J. Virol. Methods 5, 191-197). Diagnosis based on the detection of neutralizing antibodies, on the other hand, should have a high predictive value for immune status and prevention of rubella infection or reinfection cases.
These differences are important, not only in evaluating peptide-based vaccines against rubella but in assaying the immune status of patients with respect to rubella infectivity. For example, the "purified" rubella antigens now available are potentially infectious and carry both the hemagglutinating and neutralizing epitopes. Thus, specific tests for immune status using these antigens are questionable, and the antigens used in those vaccines may be infectious.
Considering these problems, we have selected certain peptide sequences on the E1 and E2 proteins of the rubella virus and prepared peptides defined by them. These peptides selected for their ability to bind high levels of antibodies, as measured by an ELISA, are useful in diagnostic tests for rubella infection. Peptides of this invention recognized by neutralizing antibodies are also useful as the active ingredient of a substantially innocuous rubella vaccine.
The E1 antigenicity is independent of its glycosylation (Ho-Terry and Cohen, 1984, Arch. Virol. 79, 139-146). The glycosyl moiety is often responsible for non specific interactions in immunoassays. Therefore the use of synthetic peptide antigens, which are not glycosylated, is attractive.
Antibodies to E2 glycoprotein are more abundant in patients with congenital rubella syndrome. In contrast, antibodies to E1 predominate in most other patients (Katow and Sugiura, 1985, J. Clin. Microbiol. 21, 449-451). Thus, each individual peptide of this invention can be used in the differential diagnosis of rubella infections.
Novel peptides and peptides mixtures are disclosed for use in the screening of blood or body fluids for prior exposure to the rubella virus and in the preparation of a safe, effective vaccine against rubella infections. Peptides of the E2 protein are surprisingly active both in diagnosis, and in stimulating protective antibodies. E1 peptides in admixture with the E2 peptides are the preferred antigens of this invention.
The peptides of this invention are useful in a wide variety of specific binding assays for the detection of antibodies to rubella virus, as immunogens for eliciting antibodies which could then be used for the detection, isolation or purification of rubella antigens. The peptides may also be used in the preparation of vaccines against rubella viral infections.